The vital phosphorylation posttranslational modification is regulated by two classes of enzymes, the kinases which attach phosphate groups to proteins and the phosphatases which remove them. Phosphorylation plays a key role in cell signalling and thereby regulates a plethora of important cellular processes, such as apoptosis, division, differentiation, etc. Since deregulation of such processes is often implicated in serious diseases, e.g. cancer, diabetes and neurodegenerative diseases, it is no wonder that both enzyme families present important targets for drug research.
Of the two classes of enzymes, kinases have by far been most widely studied and targeted therapeutically, leading to several commercial drugs, e.g. Gleevec. Even though phosphatases are just as promising from a drug development perspective, the field has advanced much less. An important reason for this difference is the fact that in the kinase field many research tools are available for activity profiling, target identification, high throughput screening, etc. which is crucial for drug development. However, for phosphatases such tools are severely lacking.
A key problem in this respect is the fact that the product of the action of phosphatases on proteins or peptides is a natural amino acid (i.e. a tyrosine, threonine or serine residue) which is too small to be recognized by an antibody in a protein sequence-independent manner and may also already occur at different places in the protein substrate leading to a high and sequence dependent background signal. In fact, up to now the desired route—detection of formation of dephosphorylated protein or peptide—has been virtually impossible since the product of the phosphatase reaction is often tyrosine which is not a selective target for antibodies and which leads to interference with tyrosine residues elsewhere in the protein substrate. Furthermore, most current techniques only allow end-point determination instead of monitoring the reaction kinetics. From such a kinetic experiment much more information can be gained in one experiment about the phosphatase of interest.
An alternative which has been explored involves monitoring consumption of starting material since a number of detection methods for phosphorylated protein or peptide residues exist, for instance using radioactive phosphate isotopes, phosphate chelators or antibodies. However, such methods are hampered by the fact that measurement of a decreasing signal is inherently significantly less sensitive than monitoring an increasing signal. Furthermore, if the phosphate group is detected by binding to an antibody or chelator, these probes will obviously compete with the phosphatase for binding to the substrate leading to difficulties in interpretation of the resulting data.
Furthermore, in contrast to the kinases, existing detection strategies have not yet led to efficient and performant high-throughput phosphatase enzymatic activity assays e.g. microarray assays, which will be of the essence for diagnostic and therapeutic exploitation of these important enzymes.
Consequently, there remains a need for methods and tools that allow the monitoring and study of phosphatase activity, particularly in a high-throughput, parallel manner. This is required to move forward in e.g. phosphatase drug development and phosphatase-based diagnostics.
The present invention aims at providing methods and tools for detecting hydrolase enzyme activity in general and more specifically phosphatase activity.